Vol.3 No.2 2010

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Research paper : Development of an accurate and cost-effective quantitative detection method for specific gene sequences (N. Noda)−170−Synthesiology - English edition Vol.3 No.2 (2010) environmental cleaning using bacteria. As covered in the newspapers and other media, the “brownfield” with soil contamination has become an issue. For cleaning such soil contamination, bioremediation using bacteria is thought to be effective in terms of cost. However, when cleaning up the contaminant by introducing the bacteria in the environment, the bioremediation guideline states that it is necessary to assess not only the introduced bacteria, but also the effect on the microbial community that originally exists in the soil. The method using the genetic information is effective for the assessment of the microbial community, and the gene quantification technology is drawing attention in this field. There is a diversity of microbes in the environment, and the microbes to be investigated change for each soil type. Therefore the universal QProbe PCR method that allows quantifying various gene sequences using one type of fluorescent probe is extremely effective in the detection and quantification of the various environmental microbes. Therefore, one of the ways of practical use for the universal QProbe PCR is the subcontracted analysis business in the field of environmental cleaning business where the various microbial community can be detected and quantified at low cost and in a short time.Since the ABC-PCR method has different advantages and disadvantages from the universal QProbe PCR, the scenario for its realization differs. The advantages of ABC-PCR are that accurate quantification is possible without the effect of the gene amplification inhibitor, and the target gene can be quantified by simply measuring the fluorescence after the gene amplification reaction. For the former, the users who are already using the RT-PCR but are facing the problem of amplification inhibitors will probably see merit in introducing this method. Moreover, in this method, the target gene can be quantified by simply measuring the fluorescence after the gene amplification reaction, and the expensive thermal cycler for RT-PCR is not necessary. Instead, the fluorescence measurement device for measuring the fluorescence after the gene amplification reaction is necessary. The marketing of the fluorescence measurement device (named EGBox) is in progress at the joint researcher J-Bio 21 Corporation (Fig. 10). This device is specialized to measure fluorescence in the ABC method. The specifications are: one fluorescence measurement area; depth 18 cm × width 30 cm × height 15 cm; 3.5 kg; LED light source; and three excitation wavelengths. The fluorescence value can be measured simply by inserting the PCR tube into the sample port. J-Bio 21 Corporation is trying to keep the retail price below 1 million yen. By offering such inexpensive fluorescence measurement device and reagent kit, we believe we can do business with people who wish to be involved in the gene quantification technology but are hampered by the facility cost. The ABC-PCR is extremely appropriate for cases that wish to introduce the gene quantification technology at low cost such as in developing countries. To realize such a scenario, the issues are downsizing to portable size, and energy-saving configuration where the device can be powered by batteries. As the technologies to achieve downsizing and energy saving, the micro total analysis system (-TAS), where reaction, separation, and detection are done in microspace, by forming the flow channels and circuits on a silicon or glass substrate using the microfabrication technology, was developed and is being used in the analysis of biomolecules such as nucleic acids and proteins. Downsizing and energy saving will be achieved by fusing the -TAS technology and the ABC method. The ABC method can be used in combination with the gene amplification methods other than the PCR. For example, if it is combined with the isothermal gene amplification method, quantification can be done with a simple and inexpensive device consisting only of an isothermal device with low energy use instead of a thermal cycler. To develop these technologies, there are many issues that must be solved such as the selection of gene amplification technologies (development of a new isothermal gene amplification method if necessary) as well as the development of simple nucleic acid extraction technologies. However, if these issues are overcome, it is expected that a gene quantification technology that is simpler and less expensive than the one currently used widely can be achieved.The dream of the author is the diffusion of the gene detection and quantification technologies based on the universal QProbe and the ABC methods, in pursuit of convenience and cost performance. We also aim for the development of the new technology combining the universal QProbe and the ABC methods in the future. Specifically, this involves the replacement of the fluorescent probe used in the ABC method with the universal QProbe, but there are several difficult problems because it is necessary to advance the idea on the joint DNA. However, the technology in which the universal QProbe and ABC methods are integrated will have both the accuracy of the ABC method and the flexibility of the universal QProbe method, and is expected to have great social impact due to its cost performance and the reduced preparation time for probe synthesis.Fig. 10 Prototype of simple fluorescence measurement device (EGBox) (made by J-Bio 21 Corporation)Sample portSpecifications: one fluorescence measurement area; depth 18 cm × width 30 cm × height 15 cm; 3.5 kg; LED light source; 3 excitation wavelengths

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